Summary Abstract Brain tumors are responsible for an immense burden of disease due to their diversity, prevalence, poor response to therapies, and high morbidity and mortality. More than half a million people in the United States have a primary brain tumor or other central nervous system tumor, and about 80,000 new tumors are diagnosed each year. However, the origins of brain tumors? i.e. the specific cell type from which each brain tumor arises? remains enigmatic and is a fundamental unknown in our understanding of their biology. This project will address this major gap by developing a novel single-cell genomics technology to identify the cells of origin of brain tumors. Animal models have shown that the phenotype of the ?cell of origin?? the first cell that acquires the genetic mutations to initiate a tumor? often plays a major role in determining subsequent tumor phenotype and behavior. The epigenetic legacy of the cell of origin is likely at play in human brain tumors as well, but the identification of tumor cells of origin has not been possible due to a major technological limitation? the absence of a method for systematically tracing lineages of cells in human tissues. We will address this research challenge via three related aims: 1) Development of a novel single-cell technology called TAPESTRY capable of reconstructing high-resolution lineage trees from human tissues for the first time. TAPESTRY will achieve this by selectively capturing spontaneously occurring somatic mutations in tens of thousands of single cells, while simultaneously profiling the transcriptomes of the same single cells, thereby allowing reconstruction of phenotypically annotated lineage trees at feasible costs; 2) Application of TAPESTRY to catalogue the hierarchy of progenitor cell lineages in normal cerebral cortex as a reference, followed by identification of the cells of origin and early lineages of individual brain tumors within that hierarchy. This aim will focus on three tumor types with likely different cells of origin: glioblastoma (the most common and most lethal malignant brain tumor), anaplastic oligodendroglioma, and primitive neuroectodermal tumors; 3) In situ spatial mapping of the early tumor lineages identified by TAPESTRY to understand whether they occupy specific domains within tumors and whether they contribute to tumor migration and invasion. Overall, this work will provide insight into a significant unknown factor in brain tumor etiology?the cell of origin?which, we hypothesize plays a major and under-appreciated role in tumor biology. Identifying the cell of origin would transform our understanding of why brain tumors arise where and when they do, their phenotypic diversity, and importantly, could facilitate earlier detection, the creation of new animal models, and the design of lineage- targeted therapies. Moreover, the technology developed here has the potential for major impact across cancer biology and developmental biology research more generally.